DESCRIPTION: Filaggrin is a differentiation-related epidermal protein that interacts with and organizes epidermal keratin filaments, modulating their architecture so as to increase the strength of the stratum corneum. A very large (nearly 1 million MW in some species) precursor, profilaggrin, initially aggregated after translation into large, insoluble keratohyalin granules , is processed by a sequence of phosphorylations, dephosphorylations, and proteolytic cleavages into a large number of active filaggrin subunits. The overall objective of the study is to obtain a molecular description of these processing events, the enzymes that catalyze them, and the mechanisms that regulate the expression and activities of these enzymes so precisely during epidermal stratum corneum formation. The study will make extensive use of an electrospray ionization mass spectrometer to identify the phosphorylation states of regions of the profilaggrin molecule and the enzyme-catalyzed cleavage sites. Previous research by the applicant has identified casein kinase 2 and a novel PKC ( PKCx ) as the major activities in epidermal extracts that can phosphorylate profilaggrin in the test tube. A candidate phosphorylase, PP2A, has also been identified. One specific aim is to purify these enzymes sufficiently to obtain partial amino acid sequences. This information will be used to obtain full-length coding sequences, if necessary to characterize them as novel proteins, and to make hybridization probes and antisense oligonucleotides to explore the mechanisms regulating their expression during epidermal differentiation and to confirm their role in filaggrin processing. The proposed studies will enhance understanding of the regulatory events that lead to keratohyalin disassembly and profilaggrin processing, and the role of filaggrin in reorganizing keratin filaments during epidermal differentiation, thereby providing a firm biochemical basis for future studies of epidermal diseases in which profilaggrin processing may be altered.